It is well documented that a great variety of pollutants can be found throughout the environment. Pollutants may be, for example, polar and non-polar compounds, detergents, and heavy metals. The accurate identification of specific types of pollutants present in a water or soil sample facilitates the determination of what, if any, danger is present, as well as the formulation of a plan for removing the pollutant or preventing its further accumulation. Thus, the ability to quickly, easily, and accurately determine whether an environmental sample (soil, sediment, water, ashes, etc.) is contaminated with a specific toxicant can be of great importance to wastewater and water treatment plant operators, hazardous waste managers, health officials, and others who have an interest in protecting public health and the environment from toxic insult. Although sophisticated techniques for analyzing environmental samples are well known, these techniques are often costly, time consuming, and cannot be done in the field because instrumentation and extensive sample preparation are necessary. Moreover, these techniques do not indicate whether the sample is toxic to the biota.
As used herein, the term toxicants refers to compounds, elements, or other entities in an environmental sample which, alone or in combination, may be injurious to humans or other living things. As used herein, the term heavy metals refers to metals such as antimony (Sb), arsenic (As), beryllium (Be), cadmium (Cd), copper (Cu), chromium (Cr), lead (Pb), mercury (Hg), nickel (Ni), selenium (Se), silver (Ag), tellurium (Te), and zinc (Zn). Existing methods for testing environmental samples for toxicants include both chemical and biological assays. Chemical assays whereby reagents are added to a test sample are well known. Chemical assays which are performed in the field often lack selectivity and provide ambiguous results. Of course, more sophisticated chemical assays can be performed in laboratories, but the sample preparation needed and expensive instrumentation limit the utility of these procedures. Recently, more attempts have also been made to develop biological toxicity assays. For example, several microbial assays have been developed for assessing chemical toxicity. These biological assays can be based on the effects of certain toxic chemicals on microorganisms. For example, toxic chemicals in a test sample may inhibit growth, respiration, motility, viability, enzyme activity or biosynthesis, bioluminescence, photosynthesis, heat production, and ATP. However, there are many obstacles which must be overcome in order to develop a biological assay that can accurately identify the presence of toxic agents. Specifically selected microbes must be found which have the desired sensitivity for toxicants. The toxicant must not only exert some type of biological effect on the microbe but, also, that effect must be easily detectable for the assay to have any utility. Finally, the effect of toxicants must be independent from any effect caused by non-toxicants. Before the current invention, no bioassay had been developed which could selectively detect the presence of specific toxicants in a sample.
Pollutants can also be in the form of biological entities such as bacteria, viruses, and protozoa. The direct detection of pathogenic bacteria and viruses and cysts of protozoan parasites requires costly and time-consuming procedures, and well-trained labor. Therefore, more easily-monitored non-pathogenic microbes which are known to be associated with pathogenic microbes are often used as "indicator organisms" of microbial pollution. Traditional bacterial indicators used to detect fecal pollution in the environment are total coliforms, fecal coliforms, and Escherichia coli. Commercially available enzymatic tests such as Colilert.TM. and Coliquik.TM. detect simultaneously, in 24 hours, both total coliforms and E. coli in environmental samples. In both assays, total coliforms are detected by observing .beta.-galactosidase activity, which is based on the hydrolysis of the substrate o-nitrophenyl .beta.-D-galactopyranoside (ONPG) to the yellow nitrophenol which absorbs light at 420 nm. E. coli detection is based on its ability to produce an enzyme, called .beta.-glucuronidase, which hydrolyses a fluorogenic substrate, 4-methylumbelliferone glucuronide (MUG) to a fluorescent end product which can be easily detected with a long-wave UV lamp. After 24-hour incubation, samples positive for total coliforms turn yellow, whereas E. coli-positive samples fluoresce under a long-wave UV illumination in the dark.